CN105505971A - Method for improving yield of butanol produced by escherichia coli - Google Patents

Method for improving yield of butanol produced by escherichia coli Download PDF

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CN105505971A
CN105505971A CN201610035150.5A CN201610035150A CN105505971A CN 105505971 A CN105505971 A CN 105505971A CN 201610035150 A CN201610035150 A CN 201610035150A CN 105505971 A CN105505971 A CN 105505971A
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butanols
mdh
bacterium
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sequence
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董红军
赵春华
张延平
李寅
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Institute of Microbiology of CAS
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Abstract

The invention discloses a method for improving yield of butanol produced by escherichia coli. According to the method for constructing recombinant bacteria, expression of a malate dehydrogenase gene (mdh) on the genome of an original strain for producing butanol is inhibited or silenced to obtain the recombinant bacteria. Experiments prove that the modified target capable of promoting escherichia coli to produce butanol is the malate dehydrogenase gene (Genbank: mdh gene (b3236) in the NC_000913.3 sequence). By a lembda-red homogenous recombinant system knockout technology, after the mdh gene of the engineering strain of escherichia coli for producing butanol is knocked out, the butanol output can be increased by 283 percent, and the yield can be increased by 89 percent.

Description

A kind of method improving intestinal bacteria production butanols
Technical field
The present invention relates to biological technical field, particularly relate to a kind of method improving intestinal bacteria production butanols.
Background technology
Butanols is a kind of important chemical, and directly can be used as the precursor of organic solvent and the multiple ester compound of synthesis, it is also a kind of than ethanol biofuel advantageously simultaneously.Biological butanol under anaerobic can be fermented through ABE (Acetone-Butanol-Ethanol) by some clostridium bacterial strains and produce, and has so far and exceedes centenary history.But due to the gram positive bacterium that clostridium bacterial strain itself is strictly anaerobic, genetic manipulation difficulty, and due to complicated metabolic regulation mechanism itself, be therefore difficult to promote the ability that clostridial fermentation produces butanols, thus limit the biological butanol market competitiveness.By means of modern biotechnology, scientist starts to utilize the pattern bacteria Escherichia coli of easy handling to produce butanols, achieves significant progress.
2008, ShotaAtsumi, first JamesC.Liao etc. achieve the synthesis (AtsumiS of butanols in intestinal bacteria, CannAF, ConnorMR, etal.MetabolicengineeringofEscherichiacolifor1-butanolpr oduction.Metabolicengineering, 2008,10 (6): 305-311.), the butanols route of synthesis in clostridium acetobutylicum body is transferred in intestinal bacteria by this study group, causes it can produce a small amount of butanols.In several years subsequently, a large amount of related works is carried out gradually and is delivered, the intestinal bacteria butanols output that current report is the highest is 14-15g/L, close to clostridium output, but sufficient advantage is not shown compared with clostridium, also reach far away the industrialization level having the market competitiveness, still need more improvement, such as output, productive rate, substrate utilization etc.Therefore also need further strain improvement, improve production of butanol performance, this just needs to find new effective transformation target spot.
Summary of the invention
The present invention's object there is provided a kind of method building recombinant bacterium.
Method provided by the invention, for suppression or the reticent malate dehydrogenase gene mdh set out on bacterium genome producing butanols express, obtains recombinant bacterium.
In aforesaid method, above-mentioned suppression or the reticent malate dehydrogenase gene mdh set out on bacterium genome producing butanols are expressed as the malate dehydrogenase gene mdh set out on bacterium genome knocking out and produce butanols.
In aforesaid method, the homologous recombination system that the above-mentioned malate dehydrogenase gene mdh set out on bacterium genome knocking out production butanols adopts λ-red homologous recombination system, sacB gene mediated screens or CRISPR/Cas system.
In aforesaid method, the above-mentioned malate dehydrogenase gene mdh set out on bacterium genome producing butanols that knocks out adopts λ-red homologous recombination system that the malate dehydrogenase gene mdh set out on bacterium genome producing butanols is replaced with the marker gene of two ends with FRT.
In aforesaid method, described marker gene is kalamycin resistance gene;
Described two ends are classified as sequence 2 41-1534 position in sequence table with the nucleotides sequence of the marker gene fragment of FRT.
In aforesaid method, the bacterium that sets out of described production butanols is intestinal bacteria, and described intestinal bacteria are the butanols route of synthesis in clostridium acetobutylicum body is transferred to the bacterium that the intestinal bacteria that set out obtain;
Described intestinal bacteria are specially EB205CGMCCNo.11985;
The described intestinal bacteria that set out are BW25113.
In aforesaid method, the aminoacid sequence of described malate dehydrogenase (malic acid dehydrogenase) is sequence 3;
The nucleotide sequence of described malate dehydrogenase (malic acid dehydrogenase) mdh gene is specifically sequence 1.
In aforesaid method, describedly replace with two ends comprise the steps:, by containing homologous recombination DNA fragmentation importing EB205 (pKD46) of two ends with the kalamycin resistance gene of FRT, to obtain recombinant bacterium with the method for the marker gene of FRT by producing the malate dehydrogenase gene mdh set out on bacterium genome of butanols;
Described EB205 (pKD46) is for import the bacterium obtained in EB205CGMCCNo.11985 by plasmid;
Describedly comprise mdh upstream region of gene homology arm, upstream FRT, kalamycin resistance gene, downstream FRT and mdh downstream of gene homology arm containing two ends with the homologous recombination DNA fragmentation of the kalamycin resistance gene of FRT;
Described is specifically sequence 2 containing two ends with the nucleotide sequence of the homologous recombination DNA fragmentation of the kalamycin resistance gene of FRT.
The recombinant bacterium prepared by aforesaid method is also the scope of protection of the invention.
Aforesaid method or above-mentioned recombinant bacterium are also the scope of protection of the invention producing the application in butanols or raising butanols output.
Another object of the present invention is to provide a kind of method of producing butanols.
Method provided by the invention, comprises the steps: the above-mentioned recombinant bacterium that ferments, obtains butanols.
Intestinal bacteria EB205 bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 11st, 2016 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.11985, and Classification And Nomenclature is colon bacillus Escherichiacoli.
Experiment of the present invention proves, can promote in the present invention that the transformation target spot that intestinal bacteria produce butanols is malate dehydrogenase gene (the mdh gene (b3236) in Genbank:NC_000913.3 sequence), technology is knocked out by λ-red homologous recombination system, produce in a strain in the colibacillus engineering strain of butanols and knock out mdh gene, butanols output can improve 283%, and yield can improve 89%.
Accompanying drawing explanation
Fig. 1 is the production of butanol approach in EB205 body.
Fig. 2 is the DNA fragmentation that pcr amplification contains the kalamycin resistance gene operon of mdh DNA homolog fragment.
Fig. 3 is the PCR checking of mdh knock out mutants body.
Fig. 4 is the result schematic diagram that EB205 △ mdh::kan fermented liquid use high performance liquid chromatography (HPLC) is analyzed.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Following embodiment intestinal bacteria are the butanols route of synthesis in clostridium acetobutylicum body is transferred to the bacterium that BW25113 obtains, and are specially EB205CGMCCNo.11985.
Intestinal bacteria EB205 bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 11st, 2016 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.11985, and Classification And Nomenclature is colon bacillus Escherichiacoli.
Embodiment 1, knock out mdh gene preparation recombinant bacterium in intestinal bacteria EB205
The following examples be that λ-red homologous recombination system carries out knocking out mdh gene, the plasmid wherein related to:
PKD4 is documented in as in Publication about Document: Datsenko, KirillA., andBarryL.Wanner.One-stepinactivationofchromosomalgenesi nEscherichiacoliK-12usingPCRproducts.ProceedingsoftheNat ionalAcademyofSciences97.12 (2000): 6640-6645;
PKD46 is documented in as in Publication about Document: Datsenko, KirillA., andBarryL.Wanner.One-stepinactivationofchromosomalgenesi nEscherichiacoliK-12usingPCRproducts.ProceedingsoftheNat ionalAcademyofSciences97.12 (2000): 6640-6645;
The nucleotides sequence of mdh gene is classified as sequence 1 in sequence table, and the aminoacid sequence of the albumen malate dehydrogenase (malic acid dehydrogenase) of its coding is sequence 3 in sequence table.
1, the preparation of recombinant bacterium EB205 (pKD46)
EB205 competent cell: the 200mlEB205 bacterium liquid being cultured to mid log phase in triangular flask, ice bath 30min.4 DEG C, the centrifugal 5min of 3000g.Discard supernatant liquor, with the resuspended thalline of aseptic 10% glycerine of precooling, wash twice.Finally add the resuspended thalline of 10% glycerine of 1ml precooling, 50 μ l often manage and point to be filled in the 1.5ml sterile centrifugation tube of precooling for subsequent use, obtain EB205 competent cell.
EB205 (pKD46) recombinant bacterium: get 1 μ lpKD46 and mix with above-mentioned EB205 competent cell, ice bath 5min, be transferred to by mixture in 2mm electric shock cup and transform, electroporated parameter is: voltage 2.5kV, 25 μ F, resistance 200 Ω.Typical case is 5ms the electric shock time length.After electricity transforms, go in 1mlLB substratum by bacterium liquid immediately, rejuvenation 1h, coat on the LB flat board containing 100 μ g/ml penbritins, 30 DEG C of aerobic cultivation 12h, obtain EB205 (pKD46) recombinant bacterium.
EB205 (pKD46) competent cell: be forwarded in the liquid LB containing 100 μ g/ml penbritins by the EB205 that the flat board of above-mentioned coating grows (pKD46) recombinant bacterium bacterium colony, 30 DEG C are cultured to OD 600when value is about 0.2, adding final concentration is that the pectinose of 10mmol/l carries out abduction delivering 2 hours, prepares EB205 (pKD46) competent cell.
Fig. 1 is the production of butanol approach in EB205 body.
2, the recombinant bacterium EB205 △ mdh::kan of mdh is knocked out
1), the preparation of mdh homologous recombination fragment
Use pKD4 is template, carrying out pcr amplification with 1032-mdh-KoF and 1032-mdh-KoR primer, obtain the pcr amplification product (Fig. 2) of about 1.6kb, is mdh homologous recombination fragment, through order-checking, the nucleotides sequence of mdh homologous recombination fragment is classified as sequence 2 in sequence table.
Mdh homologous recombination fragment comprises mdh upstream region of gene homology arm, upstream FRT, kalamycin resistance gene, downstream FRT and mdh downstream of gene homology arm; Wherein, mdh upstream region of gene homology arm is sequence 2 1-40 position, upstream FRT is sequence 2 59-106 position, kalamycin resistance gene is sequence 2 468-1262 position, downstream FRT be sequence 2 1453-1498 position and mdh downstream of gene homology arm is sequence 2 1535-1574 position.
1032-mdh-KoF:
AAAACCCAACTGCCTTCAGGTTCAGAACTCTCTCTGTATGTGTAGGCTGGAGCTGCTTC，
1033-mdh-KoR:
CGTTTTTACCCAGCAGCAGCGGTTGAGAGAAGAAACGGGCTGGGAATTAGCCATGGTCC，
In above-mentioned primer sequence, underscore part is the homologous fragment of mdh gene, and other parts are the sequences from pKD4 masterplate.
The archaeal dna polymerase that above-mentioned PCR uses is TAKARAPrimeSTARHS high-fidelity enzyme, and above-mentioned PCR program is as table 1:
Table 1 is PCR program
2), mdh is knocked out by homologous recombination
By 1 μ l above-mentioned 1) the mdh homologous recombination fragment prepared mixes with above-mentioned 1 EB205 prepared (pKD46) competent cell, transform according to the electric shock transformation method in above-mentioned 1,30 DEG C of rejuvenation 1.5h, coat on the LB flat board containing 50 μ g/ml kantlex, cultivate 24h, obtain recombinant bacterium for 37 DEG C.
Using recombinant bacterium as template, carry out pcr amplification with primer 1034-mdh-1 and 1034-mdh-2.Take EB205 as contrast.
1034-mdh-1：ATGAAAGTCGCAGTCCTCGGCGCTGCTGGC；
1035-mdh-2：GTTCTGTTCAAATGCGCTCAGGGTACCGAT，
By above-mentioned PCR primer electrophoresis detection, result is as Fig. 3, and the recombinant bacterium obtaining about 1.7kb fragment is positive recombinant bacterium, and EB205 obtains about 0.8kb fragment.
This positive recombinant bacterium called after EB205 △ mdh::kan, this bacterium is the mdh on EB205 genome is replaced with the recombinant bacterium of two ends with the kalamycin resistance gene fragment (sequence 2 41-1534 position) of FRT.
The fermentation of embodiment 2, recombinant bacterium EB205 △ mdh::kan
Recombinant bacterium EB205 △ mdh::kan embodiment 1 obtained and the toothpick of control strain EB205 sterilizing are inoculated in the 10mlM9Y substratum in the centrifuge tube of 15ml capacity, utilize toothpick to inoculate and to be placed in 37 DEG C of constant incubators standing for fermentation 2 days, obtain fermented liquid.Experiment in triplicate.
Above-mentioned M9Y substratum is by 17.1g/lNa 2hPO 412H 2o, 3.0g/lKH 2pO 4, 0.5g/lNaCl, 2.5g/lNH 4cl, 2g/lYeastExtract, 22g/lC 6h 12o 6h 2o, 2mMMgSO 47H 2o, 0.1mMCaCl 2form with water.
Get above-mentioned fermented liquid centrifugal after supernatant liquor via hole diameter size be carry out efficient liquid phase chromatographic analysis after the metre filter of 0.22 μm, analyze and adopt Agilent1260 liquid chromatograph, differential refraction detector, BioRadAminexHPX-87H organic acid post, column temperature 15 DEG C, moving phase is 5mmol/l aqueous sulfuric acid, flow velocity 0.5ml/min, sample size 10 μ l.
Standard substance glucose and butanols retention time are respectively about 10.2min and 39.8min, the peak value of time about 10.2min and 39.8min that with a hook at the end in fermented liquid supernatant liquid, therefore contain glucose and butanols in supernatant liquor.
According to the actual relationship of reference material concentration and peak area, the calculation formula determined is:
The calculation formula of glucose concn: y=194111x, R 2=0.9999, wherein x represents glucose concn (g/L), and y represents actual peak area;
The calculation formula of butanol concentration: y=138232x, R 2=0.9998, wherein x represents butanol concentration (g/L), and y represents actual peak area.
Calculate the butanols yield result of bacterial strain as shown in following table 2 and Fig. 4, containing glucose and butanols in fermented liquid supernatant liquid, compared with not knocking out the EB205 of mdh gene, knock out the butanols output increased of mdh gene recombination bacterium EB205 △ mdh::kan 283%, yield (butanols output/glucose utilization) improves 89%, proves to knock out in intestinal bacteria the production that mdh gene is conducive to butanols.
Table 2 knocks out the impact on butanols output after mdh gene
In above-mentioned glucose utilization=M9Y substratum glucose concn-fermented liquid supernatant liquor in glucose concn.

Claims (10)

1. build a method for recombinant bacterium, for suppression or the reticent malate dehydrogenase gene mdh set out on bacterium genome producing butanols express, obtain recombinant bacterium.
2. method according to claim 1, is characterized in that: described suppression or the reticent malate dehydrogenase gene mdh set out on bacterium genome producing butanols are expressed as the malate dehydrogenase gene mdh set out on bacterium genome knocking out and produce butanols.
3. method according to claim 2, is characterized in that: described in knock out the malate dehydrogenase gene mdh on bacterium genome of setting out producing butanols and adopt λ-red homologous recombination system, the homologous recombination system of sacB gene mediated screening or CRISPR/Cas system.
4. according to the method in claim 2 or 3, it is characterized in that: described in knock out and produce the malate dehydrogenase gene mdh set out on bacterium genome of butanols and adopt λ-red homologous recombination system that the malate dehydrogenase gene mdh set out on bacterium genome producing butanols is replaced with the marker gene of two ends with FRT.
5. method according to claim 4, is characterized in that: described marker gene is kalamycin resistance gene;
Described two ends are classified as sequence 2 41-1534 position in sequence table with the nucleotides sequence of the marker gene fragment of FRT.
6., according to described method arbitrary in claim 1-5, it is characterized in that:
The bacterium that sets out of described production butanols is intestinal bacteria, and described intestinal bacteria are the butanols route of synthesis in clostridium acetobutylicum body is transferred to the bacterium that the intestinal bacteria that set out obtain;
Described intestinal bacteria are specially EB205CGMCCNo.11985;
The described intestinal bacteria that set out are BW25113.
7., according to described method arbitrary in claim 1-6, it is characterized in that:
The aminoacid sequence of described malate dehydrogenase (malic acid dehydrogenase) is sequence 3;
The nucleotide sequence of described malate dehydrogenase (malic acid dehydrogenase) mdh gene is specifically sequence 1.
8. the recombinant bacterium prepared by described method arbitrary in claim 1-7.
9. in claim 1-7 recombinant bacterium described in arbitrary described method or claim 8 in the application of producing butanols or improve in butanols output.
10. produce a method for butanols, recombinant bacterium described in fermentation claim 8, obtains butanols.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112592940A (en) * 2020-12-16 2021-04-02 江西邦泰绿色生物合成生态产业园发展有限公司 Biological enzyme synthesis method of N-fluorenyl methoxycarbonyl-decyl acetaldehyde
CN116656637A (en) * 2023-07-07 2023-08-29 上海逐药科技有限公司 Variant of malate dehydrogenase

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112592940A (en) * 2020-12-16 2021-04-02 江西邦泰绿色生物合成生态产业园发展有限公司 Biological enzyme synthesis method of N-fluorenyl methoxycarbonyl-decyl acetaldehyde
CN112592940B (en) * 2020-12-16 2023-05-02 江西邦泰绿色生物合成生态产业园发展有限公司 Biological enzyme synthesis method of N- (9-fluorenylmethoxycarbonyl) -decylaminoacetaldehyde
CN116656637A (en) * 2023-07-07 2023-08-29 上海逐药科技有限公司 Variant of malate dehydrogenase
CN116656637B (en) * 2023-07-07 2024-05-14 上海逐药科技有限公司 Variant of malate dehydrogenase

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